(4m) Targeted Modulation of the Immune Response through Molecular Design

Significance: Cytokines play critical roles in immune responses by directing immune cell differentiation and function. The interleukin 4 (IL-4) cytokine is responsible for orchestrating the T helper type 2 immune response by activating and regulating lymphocytes, myeloid cells, and non-hematopoietic cells. Like most of cytokines, IL-4 can activate multiple cell types through different receptor complexes. Specifically, IL-4 signals through either type 1 receptor complexes, composed of IL-4Rα and common gamma (g_c), or type 2 receptor complexes, composed of IL-4Rα and IL-13Rα1. Since the type 1 and type 2 receptor complexes are differentially expressed on different cell subsets, IL-4 can modulate immune homeostasis through cell type-specific signaling. Due to the crucial role IL-4 plays in promoting wound-healing and mediating the humoral immune response, there is great interest in harnessing this cytokine as a therapeutic. However, the clinical potential of the natural IL-4 cytokine is limited by its instability and pleiotropic functions. Here, we designed and characterized a de novo IL-4 cytokine mimetic (denoted Neo-4), which shows outstanding thermal stability, cell-type specific stimulation and serves as a promising therapeutic candidate.

Methods: De novo computational design was used to engineer Neo-4 based on a previously published IL-2 mimetic (Neo-2), which also engages g_c, like natural IL-4¹. 11 mutations were introduced to the Neo-2 structure to generate Neo-4 that binds to the IL-4Rα and g_c subunits (Figure 1A). Bioactivities of IL-4 versus Neo-4 were compared on various lymphocyte and myeloid cell populations, using STAT6 phosphorylation or M2-like-macrophage-associated gene expression profiles as readouts. We also compared the pro-regenerative functionality of Neo-4 with that of natural IL-4 using an in vivo volumetric muscle loss model in mice. We compared the thermal stability of IL-4 and Neo-4 by heating the molecules to 95°C for various time periods and assessing functional persistence. To further probe the hyper-stability of Neo-4, we determined whether the cytokine mimetic maintained signaling activity following 3D printing into a polycaprolactone (PCL) scaffold at 120°C. Finally, we evaluated the cell type-specific bias of Neo-4 by analyzing STAT6 phosphorylation and transcriptomic activity on multiple non-hematopoietic cell types.

Results: Overall, we observed that de novo designed Neo-4 recapitulates many functions of the natural IL-4 cytokine. Neo-4 activated STAT6 signaling in multiple lymphocyte and myeloid cell lineages. Neo-4 was also found to skew macrophages towards M2-like phenotype in vitro and in vivo. Moreover, compared to natural IL-4, Neo-4 showed remarkably superior thermal stability and maintained full bioactivity after being heated to 95°C for 3 hours (Figure 1B) or being hot extruded at 120°C with PCL during 3D printing. In addition, in contrast with native IL-4, which activates both type 1 and 2 signaling pathways, Neo-4 induced biased type 1 signaling and selectively activated cells from hematopoietic lineages, such as B cells, monocytes, and macrophages (Figure 1C).

Implications: We successfully engineered a de novo cytokine mimetic denoted Neo-4, which mimics the physiological functions of IL-4 but is hyper-stable and specifically biased toward activating type 1 signaling on hematopoietic cells. Neo-4 engineering efforts showcased that computationally designed proteins can directly incorporated into sophisticated biomaterials that require heat processing, such as 3D printed scaffolds, paving the way for future integration of de novo proteins into tissue engineering platforms. Furthermore, due its cell type-specific stimulation properties, Neo-4 offers unprecedented insight into IL-4 signaling pathways, and this novel molecule has great potential to serve as a targeted therapeutic for wound healing and tissue regeneration.